Yang Juan, Bernardo-Colón Alexandra, Becerra S Patricia
Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, Bethesda, Maryland, United States.
Invest Ophthalmol Vis Sci. 2025 Sep 2;66(12):11. doi: 10.1167/iovs.66.12.11.
Lipid accumulation in the retinal pigment epithelium (RPE) contributes to cellular stress and progression of age-related macular degeneration (AMD). However, the regulation of lipid homeostasis in AMD development is not fully elucidated. The study investigates the effects of Pnpla2 deletion, a gene involved in lipid regulation, on key markers of RPE senescence and aging with potential relevance to AMD.
RPE flat mounts and retinal cryosections were analyzed from Pnpla2-/- and Pnpla2+/+ mice aged 3 months. Senescence-associated β-galactosidase (SA-β-gal) activity was assessed in flat mounts. DAPI was used to quantify RPE cells with single or multiple nuclei. Immunohistofluorescence was carried out to assess RPE tight junctions and expression of senescence and AMD markers using antibodies to zonula occludens 1 (ZO-1), phospho-histone (P-γ-H2AX), apolipoprotein E (ApoE), and high mobility group box 1 (HMGB1). Fundus imaging was acquired, and electroretinography (ERG) assessed visual function.
Pnpla2-/- RPE exhibited increased SA-β-gal activity, multinucleation of the population, and the translocation of HMGB1 from nucleus to cytoplasm, indicative of cellular senescence. Tight junctions were disrupted. The number of P-γ-H2AX-positive RPE cells increased by 50%, suggesting increased DNA damage. ApoE levels were elevated in Bruch's membrane and subretinal regions. At 3 months of age, attenuation of ERG c-wave amplitude was observed in both Pnpla2-/- and Pnpla2+/- mice. By 7 months of age, Pnpla2+/- mice exhibited continued attenuation of ERG c-wave amplitude and developed white spots.
Pnpla2 deficiency accelerates cellular features of RPE aging and generates AMD-like features. These findings underscore the importance of PNPLA2 in mitigating AMD progression and highlight its significance in retinal health and degeneration.
视网膜色素上皮（RPE）中的脂质积累会导致细胞应激以及年龄相关性黄斑变性（AMD）的进展。然而，AMD发展过程中脂质稳态的调节尚未完全阐明。本研究调查了参与脂质调节的基因Pnpla2缺失对RPE衰老和老化关键标志物的影响，这些标志物可能与AMD相关。
对3月龄的Pnpla2-/-和Pnpla2+/+小鼠的RPE平铺片和视网膜冰冻切片进行分析。在平铺片中评估衰老相关β-半乳糖苷酶（SA-β-gal）活性。使用DAPI对单核或多核的RPE细胞进行定量。进行免疫组织荧光分析，使用针对紧密连接蛋白1（ZO-1）、磷酸化组蛋白（P-γ-H2AX）、载脂蛋白E（ApoE）和高迁移率族蛋白B1（HMGB1）的抗体来评估RPE紧密连接以及衰老和AMD标志物的表达。进行眼底成像，并通过视网膜电图（ERG）评估视觉功能。
Pnpla2-/- RPE表现出SA-β-gal活性增加、细胞群体多核化以及HMGB1从细胞核向细胞质的转位，表明细胞衰老。紧密连接被破坏。P-γ-H2AX阳性RPE细胞数量增加了50%，表明DNA损伤增加。Bruch膜和视网膜下区域的ApoE水平升高。在3月龄时，Pnpla2-/-和Pnpla2+/-小鼠均观察到ERG c波振幅衰减。到7月龄时，Pnpla2+/-小鼠的ERG c波振幅持续衰减并出现白斑。
Pnpla2缺乏会加速RPE衰老的细胞特征并产生类似AMD的特征。这些发现强调了PNPLA2在减轻AMD进展中的重要性，并突出了其在视网膜健康和退化中的意义。
